Mitochondrial disease caused by point mutations in mitochondrial DNA (mtDNA) can be a formidable diagnostic challenge. Because clinical presentation is variable, multisystemic, and mimics many other neurological disorders, ruling out a mtDNA etiology is difficult in patients with suspected myopathies or encephalopathies. Screening for mtDNA mutations that cause disease would help inform clinical decision making, but such detection is difficult. In affected tissues like the pancreas, brain, heart, and skeletal muscle, the proportion of mutant mtDNA genomes may be high, >20%, but frequencies are much lower in more accessible samples like blood - sometimes as low as 1%. Mutations that cause disease are found scattered around the 16,569 bp mitochondrial genome, and although ~40 distinct mutations account for >90% of diagnosed disease, analysis of a patient's mtDNA for mutations at all those sites is costly and technically difficult. Conventional DNA sequencing is insensitive to mutations much below 25% and even the resequencing MitoChip array (Affymetrix) performs poorly for reliable detection of mutations at frequencies below 10%. We have developed a "dip and measure" platform that reliably detects mtDNA mutations at a threshold frequency of 1%. Because our technology detects DNA targets with ultra-high sensitivity, no PCR amplification of patient DNA is required. Because detection is based on hybridization, minimal sample preparation is necessary beyond solubilization. The instrumentation that implements our technology is fully automated so that once a sample is solubilized and added to wells of a standard 96- well plate, all further operations are robotic and hands-off. Our goal is to develop this technology so that mtDNA mutations are detected in 1 <l of whole blood. To do so, we need to increase sensitivity of detection a further 10-fold - from 100,000 DNA targets (i.e., 20 zeptomoles) to d 10,000 DNA molecules. Once this sensitivity is achieved, we will translate the technology to the detection of mtDNA mutations in double-stranded DNAs. Finally, we will validate protocols for the measurement of mtDNA mutations from 1 <l of whole blood. Our Specific Aims are: 1) To increase sensitivity of detection at least 10-fold to d 10,000 DNA molecules 2) To detect mtDNA mutations in double-stranded DNAs with ultra-high sensitivity 3) To measure frequencies of mtDNA mutations in 1 <l of whole blood For mtDNA genotyping to penetrate into routine clinical practice, testing must be robust, rapid, reliable, and technically simple. We will develop such technology so that genotypic testing for mtDNA mutations becomes a low cost clinical tool rather than a research endeavor. PUBLIC HEALTH RELEVANCE: Mitochondrial disease is difficult to diagnose and difficult to rule out in patients because there is no single laboratory test that is both sensitive and specific. Since mutations in mitochondrial DNA are responsible for about 50% of cases, a laboratory test for such mutations that is both reliable and low cost will have significant benefit to clinicians in diagnosing or ruling out mitochondrial disease in patients. Our research is directed to the development of a simple blood test for mitochondrial DNA mutations that will be reliable and low cost.